Morphological changes of giant mitochondria in the unicellular to multicellular phase during parthenogenesis of Ulva partita (Ulvophyceae) revealed by expression of mitochondrial targeting GFP and PEG transformation

A giant mitochondrion that branches and connects as a single mitochondrion in a cell has been observed during specific phases of the cell cycle of unicellular green algae, but has not been observed in multicellular algae. The genus Ulva is a green macroalga in which the haploid and diploid phases are isomorphic and its gametes develop parthenogenetically. The existence or absence of the giant mitochondrion, and its behavior in Ulva partita, were investigated using a parthenogenesis system. To observe the parthenogenesis of gametes and the dynamics of mitochondria by fluorescence microscopy, we developed an experimental system for culturing and observing U. partita on cover slips: gametes were suspended in 6‐well plates filled with artificial seawater, and cover slips were placed on the well bottoms. The gametes settled on the cover slips as spherical cells (1‐cell S phase). These cells grew into larger cells, losing their eyespot (1‐cell L phase), and developed into multicellular thalli. Gene introduction using the polyethylene glycol (PEG) method is available with transformation efficiencies of 9.0–15.1%. Transformation was performed using a plasmid encoding green fluorescent protein (GFP) fused to the mitochondrial targeting sequence, and mitochondria were labeled by GFP fluorescence. This revealed a string‐shaped giant mitochondrion in a cell of the 1‐cell S phase. In the 1‐cell L phase, a reticular mitochondrion was observed. After the initiation of cell division, the reticular mitochondrion was fragmented, and small oval mitochondria were observed in the 5‐cell phase.     

Fine structure of spermatozoa inAnthopharynx sacculipenis (Plathelminthes,Solenopharyngidae)

Summary The organisation of the spermatozoa ofAnthopharynx sacculipenis is described, based on electron-microscopical observations. The male gametes are fili-form in shape. They are totally enclosed by cortical microtubules and possess two free cilia. Special features are dot-like dense granules arranged in regular rows and terraced elaborations of the nuclear membrane. Such terraced elaborations are not known in any other species of flatworms whereas dot-like dense granules are described for some other taxa of the Rhabdocoela. Male gametes do not show synapomorphic correspondences between the Solenopharyngidae and the Prolecithiphora.Abbreviations ci cilia - ct cortical microtubules - db dense bodies - gl glycogen - mi mitochondrion - n nucleus - nt nuclear terraces     

The ultrastructure of the spermatozoon of the hoplonemertine,Emplectonema neesii

Summary Mature spermatozoa of the hoplonemertine, Emplectonema neesii were studied by light and electron microscopy. The spermatozoa are flagellate and motile, each gamete consisting of an elongate anterior head and a posterior flagellar tail. Three regions are identifiable in the head, the acrosome, a nuclear zone and a connecting piece containing two centrioles. The nuclear zone contains glycogen granules as well as an elongate, grooved nucleus and a large mitochondrion whose lobes interdigitate with the nuclear grooves. The flagellum has a typical 9 + 2 flagellar tubule organisation. Nemertine spermatozoan ultrastructure, as exemplified by that of E. neesii, is compared with that of platyhelminth male gametes and the supposed phylogenetic affinity of the two taxeis reexamined in the light of the results of this comparison.My thanks are due to Miss A. Harding and Miss J. Webb for collecting sexually mature E. neesii, and to Dr. R. G. Crump for laboratory facilities at the Orielton Field Centre, Pembrokeshire, Wales. The work was carried out during the tenure of a research grant from The Nuffield Foundation.     

RELATION BETWEEN THE ACROSOME REACTION AND FERTILIZATION IN THE SEA URCHIN. II. FERTILIZATION IN ACIDIFIED SEA WATER WITH EGG-WATER-TREATED SPERMATOZOA*

Fertilization of sea urchin eggs fails to occur at a pH lower than 6.5. Analytical studies on this problem were made with Hemicentrotus pulcherrimus, Anthocidaris crassispina and Pseudocentrotus depressus. If the spermatozoa have been pretreated with egg water, eggs can be fertilized even at pH 6.5 and 6.0. The acrosome reaction is inhibited at a pH lower than 6.5. Intact spermatozoa fail to adhere to the fixed eggs in acidified sea water, whereas egg-water-treated spermatozoa adhere even at pH 6.5 and 6.0. From these results we infer that the failure of fertilization at pH 6.5–6.0 is caused by non-occurrence of the acrosome reaction, and that fertilization reactions other than the acrosome reaction, such as the binding and fusion of the gametes, are not inhibited in this range of pH. At pH 5.5, the spermatozoa become inert and fertilization is inhibited or suppressed, even though egg-water-treated spermatozoa are employed.     

The spermatozoa of three species of Xenotrichulidae (Gastrotricha,Chaetonotida): the two “dünne Nebengeisseln” of spermatozoa in Heteroxenotrichula squamosa are peculiar para-acrosomal bodies

The spermatozoa of xenotrichulid gastrotrichs have been studied with the aim of supplying further characters for the phylogenetic analysis of Gastrotricha and to assess the reported biflagellarity of Heteroxenotrichula squamosa. Three species have been examined, belonging to the two hermaphroditic genera of xenotrichulids. The spermatozoa are filiform cells characterized by a scarcely condensed nucleus followed by a single mitochondrion and a flagellum with large accessory fibers. These show an obliquely striated cortex and a core containing some dense material. In Heteroxenotrichula squamosa and Xenotrichula punctata there is also a simple acrosome flanked by two para-acrosomal bodies which are curious long extracellular structures formed by a pile of electron-dense disks connected by thin threads. Xenotrichula intermedia lacks both acrosome and paraacrosomal bodies. The sperm model of xenotrichulids is very different from that of the Macrodasyida and Chaetonotida so far studied, thus supporting an isolated position of the family. The oblique striation of the tail's accessory fibers is similar in to the one period and inclination of the strated cylinder of macrodasyid gastrotrichs, thus being the only spermatological character shared by the two gastrotrich taxa.     

Ultrastructure of the gametes ofCoelomomyces dodgei Couch (Blastocladiales,Chytridiomycetes)

Summary As part of an investigation on the developmental biology ofCoelomomyces dodgei Couch (Blastocladiales, Chytridiomycetes), the ultrastructure of the male and female gametes was studied. The nucleus is central and conical in shape except for a basal spur that curves back towards the large plate-like mitochondrion. A nuclear cap of ribosomes sits on the flat anterior end of the nucleus. Approximately seven lipid globules are partially embedded in the mitochondrion and are interconnected by membrane cisternae. The lipid globules are covered by a single fenestrated microbody and a backing membrane lies between the microbody and the gamete plasma membrane. The kinetosome is at the base of the nucleus and is connected to a single, posterior, whiplash flagellum. A nonkinetosomal centriole is absent. In the peripheral cytoplasm of both mating types there is a paracrystalline body of unknown composition and function. No significant ultrastructural differences were found between the male and female gametes.     

Surrogate production of genome-edited sperm from a different subfamily by spermatogonial stem cell transplantation

The surrogate reproduction technique, such as inter-specific spermatogonial stem cells (SSCs) transplantation (SSCT), provides a powerful tool for production of gametes derived from endangered species or those with desirable traits. However, generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily, by SSCT, has not succeeded. Here, using two small cyprinid fishes from different subfamilies, Chinese rare minnow (gobiocypris rarus, for brief: Gr) and zebrafish (danio rerio), we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure. The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis, resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells. Interestingly, the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior. When pou5f3 and chd knockout Gr SSCs were transplanted, Gr-derived genome-edited sperm was successfully produced in zebrafish. This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT, and surrogate production of genome-edited gametes from another species as well. This method is feasible to be applied to future breeding of commercial fish and livestock.

     

A scanning electron microscopical study of sperm development and activation in Arenicola marina L. (Annelida: Polychaeta)

Abstract

The lugworm, Arenicola marina L. has an annual cycle of reproduction with epidemic spawning and external fertilisation. The spermatozoa of Arenicola are unusual in that they are held immotile (as plates of several hundred cells known as morulae) in the coelomic fluid until activated just prior to spawning. Activation of Arenicola sperm is brought about by a sperm maturation factor (SMF) from the prostomium and can be carried out in vitro using an assay technique developed by Bentley (1985). Scanning electron microscopy is used here to examine the changes which occur during in vitro activation. This revealed that the bundles of flagella of inactive sperm become disorganised as flagella beating commences but the flagella at this stage are still bound together at their tips. The sperm heads then become separated from the cytophore and finally the distal binding of the flagella is broken to give free-swimming spermatozoa. Coelomocytes present in the coelomic fluid resorb unspawned gametes prior to the initiation of the next gametogenic phase.     

Multicolor fluorescence in situ hybridization analysis of the spermatozoa of a male heterozygous for a reciprocal translocation t(11;22)(q23;q11)

A reciprocal translocation between chromosomes 11 and 22 is a site-specific translocation that has been seen in many families with no common ancestry. This translocation is of particular interest because balanced carriers have a 0.7–3.7% risk of having children with the supernumerary der(22), resulting from a 3:1 segregation. We have used a three color fluorescence in situ hybridization (FISH) with specific DNA probes to determine the chromosome segregation pattern of a male carrier of a translocation t(11;22)(q23;q11). The probes selected included a centromeric marker for chromosome 11, a marker closely linked to the centromere of chromosome 22, and a third probe distal to the translocation breakpoint of chromosome 22. The results showed that 3 : 1 segregation is preferential in this patient, with 40.1% of spermatozoa belonging to this segregation type. Alternate segregation followed with 27.4% of analyzed spermatozoa; 17.6% resulted from adjacent 1 and 12.5% resulted from adjacent 2 segregation. We detected 0.5% of presumably diploid spermatozoa. Complementary adjacent 1 products were observed at statistically different frequencies (P = 0.02). Complementary adjacent 2 products without recombination in the interstitial segments were also seen at different frequencies (P = 0.002). In 3 : 1 segregation, the products containing one chromosome were observed more frequently than those with three chromosomes (P = 0.0001). The 24,+der(22) gamete was seen more frequently than all of the other gametes combined which had 24 chromosomes resulting from 3 : 1 segregation. The results of this study demonstrate that in this t(11;22) carrier, 3 : 1 segregation is preferential but not exclusive. Received: 9 December 1998 / Accepted: 1 March 1999     

Ultrastructure of spermiogenesis and spermatozoa in Bradynectes sterreri and remarks on sperm cells in Haplopharynx rostratus and Paromalostomum fusculum: phylogenetic implications for the Macrostomorpha (Plathelminthes)

 The fine structure of spermiogenesis and spermatozoa in three species of the Macrostomorpha was studied, with emphasis on Bradynectes sterreri. Two centrioles appear during the development of sperm cells, at least in B. sterreri and Paromalostomum fusculum. Initially these organelles have a perpendicular position, but later they come to lie in line with each other. In P. fusculum, the differentiation of rootlet structures inserting on both centrioles was found. However, ciliary axonemes do not grow out, either in B. sterreri or in P. fusculum. These two species, and also Haplopharynx rostratus, have aciliated spermatozoa. The mature male gametes of B. sterreri are characterized by a filiform nucleus, numerous mitochondria, dense bodies irregular in shape, membranous lacunae, a pair of electron-dense lateral ledges and two sets of cortical microtubules in addition to a closed ring of microtubules in the posterior segment of the cell. Both lateral ledges do not originate from the centrioles. ’Lateral ledges’ or ’lateral bristles’ were not observed in spermatozoa of H. rostratus and P. fusculum. Such structures cannot be considered autapomorphic for the Macrostomorpha. The known spermatological characteristics contribute to elucidating the interrelationships of the Macrostomorpha. Haplopharynx and Macrostomida are sister groups. Spermatozoa with cortical microtubules separated into two sets are hypothesized as an autapomorphy of the Macrostomida. The two lateral ledges found in spermatozoa of B. sterreri are discussed to correspond to the pair of ’lateral bristles’ known from Macrostomum species, indicating a sister-group relationship of these two taxa. Apparently, the aciliated spermatozoa of Macrostomorpha species originated from biciliated male gametes. Hence, biciliated spermatozoa are not an evolutionary novelty of the Trepaxonemata, but of the Rhabditophora. Accepted: 22 February 1999     

ULTRASTRUCTURE OF MALE GAMETES OF SPHAEROPLEA ROBUSTA (CHLOROPHYCEAE)1

Male gametes have been studied in Sphaeroplea robusta (Chlorophyceae) using both light and electron microscopy. Mature gametes are typically biflagellate and possess a single, large mitochondrion that dominates the anterior third of the cell, directly posterior to the basal bodies. One or more microbodies are closely associated with the mitochondrion. Contractile vacuoles occur anterior to the elongated nucleus which, in fully mature gametes, possesses condensed chromatin. The reduced, starch-filled chloroplast lacks an associated eyespot and occupies a posterior position. A thin, anteriorly directed process or extension of the chloroplast parallels a portion of one of the multistranded flagellar roots. The paired basal bodies are directly opposed with no demonstrable offset, and are connected by an arched distal fiber with a highly elaborated central striated region that forms the apical cone, a feature characteristic of male gametes in most species of Sphaeroplea. Possession of a striated distal fiber, a cruciate flagellar root system (i.e. two-stranded microtubular roots alternating with multistranded roots), and directly opposed basal bodies are consistent with the alga's chlorophycean affinities.     

Sperm ultrastructure of a member of the black coral family Aphanipathidae: Rhipidipathes reticulata (Anthozoa, Antipatharia)

Fertile male polyps of three colonies of the black coral Rhipidipathes reticulata (Aphanipathidae) from Togian Islands (Indonesia) have been the source of the sperm investigated at ultrastructural level, in order to compare their organization with that of other members belonging to the family Antipathidae and Myriopathidae. The extension of the studies to a representative of the family Aphanipathidae stresses once more the structural similarity of the male gametes in antipatharians. A sketch of the sperm model reports the similarity and differences in the examined taxa. Among the micro-characters, the cup-like body linked to the pericentriolar apparatus, is exclusive of the antipatharians. Other inclusions concern the more common pro-acrosomal vesicles or the acrosomelike structure observed only in Antipathella subpinnata and Myriopathes japonica. Lipid vesicles are occasionally present. A typical inclusion, the electron-dense content of which has a C-shaped configuration, is restricted to Rhipidipathes reticulata and is associated to the cup-like body or to the mitochondrion.     

Ultrastructure of spermiogenesis in Liza aurata Risso, 1810 (Teleostei,Mugilidae)

Summary Sequential cytological events in late spermatogenesis of Liza aurata were studied from the stage secondary spermatocytes to that of mature spermatozoa. Spermiogenesis involves preparatory morphological events followed by conspicuous modifications such as intracellular movements (diplosome and mitochondrion migration, spermatid lengthening, nuclear rotation) and structural changes (dense chromatin granules, increase in size of mitochondria, loss of cytoplasm). Mature spermatozoa, with a round nucleus and a pseudo-midpiece are of a primitive type, even compared with spermatozoa of other teleosts.     

Spermatozoon Ultrastructure in Two Species of Amphibolus (Eutardigrada,Eohypsibiidae)

We examined the ultrastructure of the spermatozoa from two species of eutardigrades, gonochoristic Amphibolus volubilis and hermaphroditic A. weglarskae, by scanning and transmission electron microscopy. The gametes from the two species were morphologically quite similar, each consisting of a short head, neck and tail. The head included a conic, corkscrew-shaped, bilayered acrosome and a cylindrical nucleus with condensed chromatin. The nucleus is surrounded by cytoplasm organized in ovoid elements with an electron-dense core. The neck is very simple, containing a centriole and unmodified mitochondria. The flagellum contains a 9+2 axoneme and terminates in a tuft of between eight and 10 microtubules. The spermatozoa of Amphibolus, like those of the other eutardigrades, are of the modified type, but nonetheless maintain some primitive aspects of the gametes from heterotardigrades.     

Gametes alter the oviductal secretory proteome

The mammalian oviduct provides an optimal environment for the maturation of gametes, fertilization, and early embryonic development. Secretory cells lining the lumen of the mammalian oviduct synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. We hypothesized that the presence of gametes in the oviduct alters the oviductal secretory proteomic profile. We used a combination of two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry to identify oviductal protein secretions that were altered in response to the presence of gametes in the oviduct. The oviductal response to spermatozoa was different from its response to oocytes as verified by Western blotting. The presence of spermatozoa or oocytes in the oviduct altered the secretion of specific proteins. Most of these proteins are known to have an influence on gamete maturation, viability, and function, and there is evidence to suggest these proteins may prepare the oviductal environment for arrival of the zygote. Our findings suggest the presence of a gamete recognition system within the oviduct capable of distinguishing between spermatozoa and oocytes.     

Apicoplast and mitochondrion in gametocytogenesis of Plasmodium falciparum

Live cell imaging of human malaria parasites Plasmodium falciparum during gametocytogenesis revealed that the apicoplast does not grow, whereas the mitochondrion undergoes remarkable morphological development. A close connection of the two organelles is consistently maintained. The apicoplast and mitochondrion are not components of the male gametes, suggesting maternal inheritance.     

Effects of cryoprotectant agents and freezing protocol on motility of black-lip pearl oyster (Pinctada margaritifera L.) spermatozoa

Gamete cryopreservation techniques have been applied to several bivalve mollusc species. However, research activity in this area has primarily focused on cryopreserving gametes from edible oysters (Ostreiidae). Few studies have examined the effect of cryoprotectants and freezing protocols in the preservation of spermatozoa from cultured pearl oysters (Pteriidae). Pearl oyster producers are increasingly looking towards the development of improved family lines and, as a consequence, the ability to cryopreserve gametes would bring about significant benefits to the cultured pearl industry. In response to this need, we evaluated the effect of three cryoprotectant additives (CPA) on motility of spermatozoa from the black-lip pearl oyster, Pinctada margaritifera. These additives have previously been used to cryopreserve gametes of other bivalve species. The following CPA mixtures were evaluated: (1) 0.45M trehalose and 0, 0.64, 1.02 and 1.53 M dimethyl sulfoxide (Me(2)SO); (2) 0.2M glucose and 2M Me(2)SO and (3) 1.31 M propylene glycol (PG). The effects of four different freezing protocols on motility of P. margaritifera spermatozoa were also evaluated (slow, medium, medium-rapid and rapid cooling). This study showed that total motility was best retained when spermatozoa were cryopreserved in 0.45 M trehalose and 0, 0.64, 1.02 or 1.53 M Me(2)SO and frozen using slow to medium-rapid cooling rates (2.1-5.2 degrees Cmin(-1)). Rapid freezing through direct plunging of spermatozoa into liquid nitrogen resulted in the lowest overall retention of motility regardless of the CPA additive; however, CPA mixture also influenced retention of motility, with 0.2M glucose in 2M Me(2)SO and 1.31 M PG retaining the lowest levels of motility for the CPAs evaluated.     

Gonad Morphology, Colony Demography and Evidence for Hermaphroditism in Gobiodon Okinawae (Teleostei, Gobiidae)

Gobiodon okinawae is a small-sized, obligate coral-dwelling goby. The majority of species in this Indo-Pacific genus exhibit an obligatory association with branching live corals and a number have been shown to be bi-directional hermaphrodites in which adults have the capacity to shift between male and female function. Gonadal histology in G. okinawae demonstrated that all individuals had either an ovotestis or an ovary. Some females had an ovary with vitellogenic oocytes, indicating adult female function. Hermaphrodites having an ovotestis were either functionally male, as evidenced by the presence of spermatozoa or, in one instance, functionally female so judged by the presence of vitellogenic oocytes. No individuals were found to have mature gametes of both sexes, although a number of hermaphrodites had an inactive ovotestis with no mature gametes of either sex. Based on size ranges for fish having different gonad morphologies, individuals in this species appear to develop first as a female. They then either mature and function solely as a female, or become hermaphroditic. Findings based on experimentally maintained individuals suggests that adult hermaphrodites can shift sexual function in either direction. Accessory gonadal structures (AGS) were found in both sexes. These consisted of two fully differentiated pairs of lobes, each compartmentalized internally by lumina and containing PAS+secretions. The secretions of the dorsally located AGS were colloidal while those of the ventrally located AGS were fibrillar and granular in nature. The presence of two sets of AGS among males appears to be characteristic of both Gobiodon and its putative sister genus, Paragobiodon. However, the occurrence of fully developed AGS in both females and males has not previously been reported and may be unique to G. okinawae.     

Spermiogenesis and spermatozoa ultrastructure of Aonides oxycephala (Annelida: Spionidae) from the Sea of Japan

Adults of Aonides oxycephala, common inhabitants of shallow boreal waters in the Atlantic and Pacific Oceans, release gametes into the water where fertilization and lecithotrophic larval development occur. During spermiogenesis, the acrosomal vesicle migrates from the posterior to the anterior end of the spermatid and the number of mitochondria reduces from six in early spermatids to four in mature spermatozoa. Each spermatozoon has an ovoid head with the acrosome 1.4?±?0.1?µm long and 1.6?±?0.1?µm wide and the nucleus 1.7?±?0.1?µm long and 2.3?±?0.1?µm in diameter, four spherical mitochondria, two centrioles oriented perpendicular to each other, putative glycogen in the shape of dense granules in the midpiece, and a flagellum with 9?×?2?+?2 organization of microtubules. The acrosome is a complex heterogeneous structure with five ordered layers of different electron densities, lying in a shallow depression on the anterior end of the nucleus. The nucleus is barrel-shaped (truncated ovoid) with the centriolar fossa housing the distal and proximal centrioles. Spermiogenesis and ultrastructure of spermatozoa of A. oxycephala are similar to those of another free spawning spionid, Marenzelleria viridis. Aonides and Marenzelleria have not, however, been considered as closely related taxa; thus, similarity in the morphology of their sperm might result from convergence or parallelism.